A longstanding mystery about the genetic messages carried by sperm led researchers to investigate an unexpected component of seminal fluid and its possible role in early embryonic development.
Study: Amyloid-mediated RNA uptake by sperm for embryonic delivery. Image Credit: Anusorn Nakdee / Shutterstock
A recent study published in the journal Molecular Cell suggests that amyloid fibrils in seminal fluid (SF) may facilitate the uptake of extracellular ribonucleic acid (RNA) by mammalian sperm and its delivery to the zygote during fertilization. Experiments using synthetic fibrils also demonstrated RNA translation in mouse embryos, suggesting a possible approach to nonviral RNA delivery during fertilization.
Previous research suggests that sperm RNA may contribute to embryonic development. Diet, stress, and environmental exposures have also been linked to changes in sperm RNA that could influence the next generation. For this transfer to occur, RNA in SF must reach sperm and remain available for delivery after fertilization. Understanding how this happens could shed light on one route through which environmental influences may affect early embryonic development.
About the study
In the present study, the researchers used cryo-electron tomography (cryo-ET) and volume electron microscopy (volume EM) along with live-cell fluorescence imaging to investigate RNA transfer from SF to sperm via amyloid fibrils. Biophysical assays, murine in vitro fertilization (IVF) experiments, RNA sequencing, and mass spectrometry (MS) were used to examine fibril transfer to embryos and analyze proteins in sperm and embryos.
The researchers used a synthetic human prostatic acid phosphatase fragment, PAP(248–286), together with its bovine and mouse homologs, to study amyloid formation. They also investigated whether PAP could undergo liquid-liquid phase separation (LLPS), given its intrinsically disordered regions (IDRs), and used RNase A to assess RNA's role in this process. LLPS occurs when molecules concentrate into distinct liquid droplets. Confocal microscopy assessed PAP-RNA colocalization, while photobleaching experiments evaluated condensate liquidity. Kinetic assays and light microscopy were used to track liquid-to-solid phase transitions.
To assess the role of sperm surface proteins in fibril uptake, human sperm cells were briefly treated with trypsin before being incubated with PAP-containing fibrils. Fluorescence-activated cell sorting (FACS) was used to assess fibril uptake in these cells. In separate experiments using bovine sperm, cryo-correlative light and electron microscopy (cryo-CLEM) and high-pressure freezing-freeze substitution serial-section electron tomography (HPF-FS ssET) were performed to assess fibril internalization.
Results
In mouse IVF experiments, PAP fibrils increased delivery of isotopically labeled HIV-2 TAR RNA to two-cell-stage embryos. When sperm were exposed to enhanced green fluorescent protein (EGFP) mRNA with PAP fibrils, the mass-spectrometry signal for EGFP protein in embryos was at least tenfold higher with fibrils than without them, indicating that transferred RNA remained available for translation.
RNA drove the PAP fragment to phase separate and form amyloid fibrils. The kinetic assays demonstrated that RNA shortened the lag phase and accelerated the transition from monomers to fibrils. Electrophoretic mobility shift assays (EMSA) provided evidence of RNA binding to the fibrillar structures formed by PAP. Cryo-electron microscopy (cryo-EM) of purified detergent-insoluble fractions from human SF identified thin fibrils measuring approximately 70 Å across, with a 4.7 Å axial rise, a structural feature characteristic of amyloid fibrils. A dot blot using a conformation-specific IgM antibody provided additional evidence of amyloid structures in the isolated fractions.
PAP-RNA phase separation persisted in 50% (v/v) human seminal plasma and in 100–200 mM NaCl. At physiological peptide concentrations of up to 60 μM, only a few condensates formed. Higher salt concentrations and RNase A treatment of polyU and polyC RNA mixtures reduced phase separation. Both bovine and mouse PAP also phase separated with RNA, indicating conserved interactions across species. These interactions may explain how RNA associates with amyloid fibrils in seminal fluid.
PAP and RNA colocalized in liquid droplets, which grew larger as PAP or RNA concentrations increased. RNA length also influenced the kinetics of amyloid fibril formation. Thioflavin T (ThT) fluorescence appeared in droplet aggregates after 32–48 hours and plateaued by 72 hours, indicating mature amyloid fibril formation. Cryo-EM revealed predominantly flat, doublet fibrils in synthetic PAP preparations, while polyU RNA accelerated fibril formation. The findings suggest that RNA-induced phase separation precedes PAP amyloid fibril formation.
Specialized RNA sequencing (PANDORA-seq) revealed that transfer RNA (tRNA)-derived fragments predominated among annotated small RNAs in amyloid-enriched fractions isolated from human SF. Among annotated RNAs measuring 35–40 nucleotides, most were tRNA-derived. Cryo-ET revealed that mammalian sperm took up synthetic PAP amyloid fibrils.
The fibrils entered bovine sperm through the midpiece within five minutes, with greater internalization and accumulation inside the cells by 45 minutes. Trypsin treatment reduced the proportion of live human sperm with internalized fibrils from 2.44% to 0.30%, suggesting that uptake depends on surface proteins.
The authors cautioned that differences between fluorescence imaging and flow cytometry made it difficult to determine the uptake rate. Direct imaging detected amyloid fibrils in about one-fifth of two-cell-stage embryos produced using sperm exposed to them before fertilization. In vitro, clusterin promoted RNA release from synthetic PAP fibrils. Molecular chaperones, including heat shock cognate 70 (Hsc70) and clusterin, were also identified in the insoluble fraction of human SF.
Implications
The findings suggest that functional amyloid fibrils in seminal fluid can bind RNA, potentially protect it from degradation, facilitate its uptake by sperm, and deliver it to the zygote during fertilization. In vitro experiments suggest that clusterin may regulate RNA release, while PAP fibrils appear to bind diverse RNA species through predominantly nonspecific electrostatic interactions.
SF amyloid fibrils could be investigated as a potential non-viral vehicle for delivering therapeutic genetic material during fertilization, particularly for families at high risk of transmitting genetic disorders. Still, this application remains speculative, and concerns about immune responses and dose-related toxicity with adeno-associated virus (AAV)-based therapies highlight the need to evaluate alternative delivery strategies.
Earlier research also found that seminal amyloid fibrils can increase HIV infectivity, so their safety requires careful evaluation. The functional delivery experiments used synthetic fibrils and exogenous RNA; transfer of naturally occurring seminal RNA, gene editing, and disease prevention were not tested.
Further studies should establish how RNA enters sperm, determine its intracellular fate, and assess whether naturally occurring amyloid-associated seminal RNA influences early embryonic gene regulation through translational control or gene silencing.
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Journal reference:
Like father, like son? Study reveals a surprising pattern in male reproductive health